V(D)J recombination and immunoglobulin heavy (IgH) class switch recombination (CSR) are the only two physiologic site-specific recombination systems in human somatic cells. These are two specialized chromosome cleavage processes that are followed by DNA joining by non-homologous end joining (NHEJ) to generate a new chromosome configuration for the benefit of the immune system. V(D)J recombination is defective in human Severe Combined Immunodeficiency (SCID), a genetic disorder that disturbs development of functional T-cells and B-cells. Recombination-activating gene (RAG) and Artemis mutations account for over one-third of human SCID patients. Knowledge of how the RAG proteins function will influence the choice of gene correction approaches in the cells from children with this form of SCID. Purified RAG proteins have been studied in their truncated forms (called core RAGs) since 1995, and this truncation removes what is called the non-core portion of RAG1 and 2. However, many RAG mutations in human SCID occur in the non-core portions. The Lieber Lab made a major breakthrough in the V(D)J recombination field by generating the first completely full-length RAG protein complexes with consistently high enzymatic activity. We also demonstrated how RAG enzymatic behavior is stimulated by interaction with active histone tails. In the preliminary data for this project, we have developed complete set of fluorescently tagged versions of our full-length RAG proteins and shown that these are still highly enzymatically active. This major advance enables recording of full-length RAG action at the single molecule level for the first time using single-molecule Forster (fluorescence) resonance energy transfer (sm-FRET), as well as bulk solution (ensemble) FRET (en-FRET). This permits us to obtain clear data on many key steps in RAG function that are not obtainable using any other methods. Combining sm-FRET with other methods that we have undertaken, such as enzyme kinetics and cryo-EM, we are in a unique position to answer many central questions for how mutations in the RAG proteins cause human SCID. This knowledge can influence how gene correction or other therapies might best be developed for SCID patients in the future. Additionally, this research is relevant to other areas, such as T-cell lymphoma chromosomal translocations involving off-target cryptic RSS sites. Similar to V(D)J recombination, lack of IgH CSR results in an immune deficiency called hyper IgM syndrome, resulting in early childhood death due to infection. In mammalian cells, IgH CSR is targeted by formation of R-loops at switch regions. Our lab has led the field in understanding R-loop formation; we recently discovered that topological tension is a key factor in affecting R-loop formation, a facet explored here more deeply. Work by others has suggested R-loop formation at other locations in the genome, which we wish to examine more carefully because it is relevant to off-target chromosome breakage during IgH CSR in human B cell lymphomas. Unification of this work under a GM R35 would synergize the work immensely.